Project Summary/Abstract There are around 50 known viruses in the Arenavirus family. These include Lassa (LASV), Lujo (LUJV), Machupo (MACV), and Junn (JUNV), which cause hemorrhagic fever in Africa and South America. The prototypic arenavirus, lymphocytic choriomeningitis virus (LCMV), also causes encephalitis and birth defects in humans worldwide. Arenaviruses express one glycoprotein on their surface, termed GPC, which is responsible for receptor engagement, cell tropism, and entry. Understanding the structure of GPC, particularly in its trimeric, prefusion conformation, is key to understanding why the different arenaviruses recognize distinct receptors and in designing therapeutics and vaccines against them. However, the GPC is metastable: it easily disassembles into its component subunits and then springs into its more stable, post-fusion conformation. This metastability hindered structural biology efforts for years. Indeed, there has been no structure of any trimeric, prefusion arenavirus GP until this year. A ten-year protein engineering effort in the lab recently culminated in the crystal structure of LASV GP in its trimeric, prefusion complex, the first such structure for any arenavirus. This landmark structure illuminated new findings: that the properly assembled prefusion trimer is essential for recognition by the most effective neutralizing antibodies and for recognition by the LASV and LCMV cell surface receptor matriglycan. In the absence of stably engineered prefusion GP, such potently protective antibodies would have been difficult to identify and characterize. There remains, however, no trimeric prefusion GP structure for any other arenavirus. Fortunately, the model built for LASV GP provides the blueprints we need to propel this effort forward. The premise of this proposal is that the correct quaternary assembly of GP can be engineered for other arenaviruses using the LASV GP structure as a template and the foundation of knowledge laid by this structure. We will combine strong preliminary results and state-of the-art biophysical techniques, with bio-layer interferometry and ELISA to analyze the interaction of engineered MACV, JUNV, LCMV and LUJV GPs with their distinct receptors and with unique panels of antibodies from human survivors (JUNV and LUJV) and mice (MACV and LCMV). In aim 1 we will develop the stable prefusion GPs necessary to determine if MACV and JUNV, like LASV, also elicit quaternary-epitope antibodies and the stoichiometry by which these viruses bind their TfR1 receptor. In aim 2 we will develop the GPs necessary to solve an LCMV-matriglycan complex, map the epitopes of LCMV-binding antibodies and identify and characterize LUJV GP antibodies and the LUJV GP structure involved in its atypical mechanism of entry. Multiple lines of inquiry will be launched by the innovative research effort proposed here.